1. Field of the Invention
The present invention relates to the methods of treatment of patients with heart disorders and more particularly to the method of using Fructose-1,6-Diphosphate in treatment of the above mentioned diseases, and also as a protective agent against unforeseen catastrophic hypotension or hypoxia during operative procedures, and as a preservative agent for transplantation organs.
2. General Background and Prior Art
In medicine and physiology it is well-known that a continuous supply of energy is necessary for the function and maintenance of a living state by cells. The degree of intracellular energy is measured by the ratio of high energy phosphate compounds to those of less energy potential (i.e., adenosine triphosphate to adenosine diphosphate and adenosine monophosphate). The biochemical pathways which produce high energy phosphate compounds have been well established in the scientific literature as a chain of reactions that result in the breakdown of the major substrates, glucose or other sugars to pyruvic and lactic acids and is a process of carbohydrate metabolism. Although one stage of glycolysis requires oxidation by dehydrogenation, this may be accomplished without oxygen, so the process as a whole may be anaerobic. The pyruvic acid formed by glycolysis is then oxidized to carbon dioxide and water. This oxidation is the source of most of the utilizable energy (ATP) derived from carbohydrate metabolism. Glycolysis also yields some energy in the form of ATP which can be utilized for muscle contraction and other functions. This is particularly important during sudden strenuous exercise, when energy must be made available in excess of that which can be provided by oxidation processes.
The glycolytic process taking place in animal tissues involves the sequence of intermediates: ##STR1##
Fructose-1,6-diP is cleared by the enzyme aldolase between the third and fourth carbon atom to form two triose phosphate molecules, glyceraldehyde-3-P and dihydroxyacetone phosphate. ##STR2##
This reaction is reversible. Glyceraldehyde-3-P and dihydroxyacetone-P are freely interconvertible through the action of triose-P-isomerase.
The next step in the main stream of glycolysis consists of the combined phosphorylation and oxidation of glyceraldehyde-3-P to 1,3 diphosphoglyceric acid, which is catalyzed by the enzyme glyceraldehyde-3-P dehydrogenase: EQU Glyceraldehyde-3-P+Pi+DPN+.revreaction.1,3 Diphosphoglyceric acid+DPN.N+H.sup.+
The conversion of glyceraldehyde-3-P to 1,3 diphosphoglyceric acid proceeds anaerobically through oxidation by DPN.sup.+. In this process DPN.sup.+ is converted to DPN.H, and the reaction would soon cease without a mechanism to reoxide DPN.H to DPN.sup.+, since the amount of coenzyme present is very small. Anaerobically DPN.H is oxidized to DPN.sup.+ by pyruvic acid, with the formation of lactic acid: ##STR3##
Substances other than pyruvic acid may serve also to oxidize DPN.H to DPN.sup.+. Among these are dihydroxyacetone-P, which is reduced to .alpha.-glycerophosphate, and oxaloacetic acid, which is reduced to malic acid. Reduction by these substances is of importance in starting the glycolytic process before sufficient pyruvic acid has been formed to function in the regeneration of DPN.sup.+. No ATP is formed in the oxidation of DPN.H by pyruvic acid.
When the supply of oxygen to the tissues and the oxidative mechanisms are adequate, the DPN.H is oxidized to DPN.sup.+ through the mitochondrial electron transport chain: EQU DPN.H-FP-Cytochromes-O.sub.2 .fwdarw.DPN.sup.+ +H.sub.2 O+3ATP
Consequently, lactic acid accumulates in tissues only when oxidation by O.sub.2 cannot keep up with glycolytic reactions and pyruvic acid is reduced to lactic acid.
In this stage of glycolysis we have the first generation of utilizable energy as ATP. 1 molecule of ATP per triose phosphate mol when DPN.H is oxidized anaerobically (by pyruvate), and 4 mols per triose phosphate mol when DPN.H is oxidized by O.sub.2.
The next stage of glycolysis consists in the conversion of 3-phosphoglyceric acid to 2-phosphoglyceric acid by the enzyme phosphoglyceromutase, which requires catalytic amounts of 2,3-diphosphoglyceric acid.
Glycolysis proceeds by the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid through action of the enzyme enolase. This reaction involves dehydration of 2-phosphoglyceric acid and is freely reversible. The loss of water converts the low-energy phosphate group of 2-phosphoglyceric acid to the high energy phosphate group of phosphopyruvic acid. Because of the high energy phosphate group present in phosphopyruvic acid, it reacts with ADP to form ATP and pyruvic acid to complete glycolysis properly. The reaction is catalyzed by the enzyme ATP-phosphopyruvic transphosphorylase or pyruvic kinase, which requires Mg.sup.++ and K.sup.+ for activation. This reaction accounts for the formation of 2 mols of ATP per mol of sugar glycolyzed.
The reactions of glycolysis in animal tissues lead to the end products pyruvic and lactic acids. Pyruvic acid is oxidized and converted to acetyl CoA by an oxidative .alpha.-ketodecarboxylase enzyme. Oxidation of the DPN.H in the electron transport chain yields 3ATP per mol. The acetyl CoA formed from pyruvic acid is oxidized in the citric acid cycle to CO.sub.2 and H.sub.2 O with the formation of 12ATP per mol. So the aerobic pathway for the breakdown of sugars and fatty acids is dependent upon a ready supply of oxygen, which is provided to the body tissues through the bloodstream and is bound weakly to hemoglobin. Thus, interruption of either the pumping action of the heart occlusion of one of the arteries or failure to oxygenate the blood being circulated by the lungs will result in either a regional or generalized unavailability of oxygen.
If oxygen is not supplied to living tissue for any of the above mentioned reasons, aerobic or oxygen dependent metabolism ceases. This leads to an attempt to compensate the failure in oxygen supply by an increase in the rate of anaerobic metabolism.
It was already noted that the anaerobic metabolic pathway for carbohydrates involves glucose which then becomes phosphorated (has six-carbon sugar). These molecules then break down to trioses (three-carbon sugars) and enter the aerobic pathway as the pyruvate molecule. In tissues that have limited blood supply or, for some other reason fail to be given an adequate amount of oxygen, the aerobic pathway must provide all of the energy necessary for cellular function. However, during any form of oxygen deprivation, whether it is from heart attack or from blood loss, leading to hypoperfusion as in an injury, the pathways of metabolism are refractory to the further entrance of the glucose into the cell, and the critical breakdown point in the metabolic pathway is at the phospho-fructo-kinase enzyme step. This means that unless something different is done, the individual deteriorates to the point where his tissues are unable, because of the injury, to regain function which may lead to a fatal outcome.
Investigations have been made on the effect of sugars on the recovery of heart functions. For example, Dr. Pasi Kettunen of Finland described his experiments on the "Comparison of the Effect of Glucose and Fructose on the Recovery of the Heart Preparation" (Scand. j. clin. Lab. Invest. 37, 705-708, 1977). Potassium citrate solution was used for heart arrest, and heart function was recovered by infusion of Locke's solution, plus glucose, fructose or sucrose. During recovery period, the amplitude and frequency of heart beats, the lactic acid in the drained perfusion solution, pH and potassium concentration were measured. The use of glucose, fructose or sucrose made no significant difference to any of these parameters. Next, the metabolism of glucose and fructose in the heart was investigated and on a metabolic basis the use of glucose for resuscitation would seem to be more appropriate than fructose.
Dr. L. H. Opie and P. Owen in their investigation of glycolysis in acute experimental myocardial infarction found out that glycolysis during anaerobic circumstances may be accelerated by all the factors that stimulate phosphofructokinase activity. They indicated that there is an overwhelming change that may be expected to inhibit phosphofructokinase activity, namely the intracellular accumulation of hydrogen ions. This phenomena was confirmed by Ui in 1960 and Kubler and Spieckermann in 1970. Dr. Opie then wrote that some index of phosphofructokinase activity can be obtained by measurements of tissue contents of glucose-6-diphosphate. Glycolytic flow may be compared to a regular stream, phosphofructokinase acts as a am-wall, inhibiting the flow of glycolysis. Thus it is evident that these investigations, being valuable by themselves could not provide a sufficient method and an agent which can modify energy requirements intracellularly in the fact of low oxygen levels, poor blood circulation or poor distribution of circulation.
3. General Discussion of the Present Invention
As it has been mentioned above, the biological attempt to compensate for insufficiency of aerobic metabolism leads to a temporary increase in the rate of anaerobic metabolism, but this compensatory mechanism is limited by acidosis of the involved tissues. Once the inevitable acidosis occurs, this pathway is inactivated also. The premature shutdown of the anaerobic pathway is not an irreversible phenomena, rather the effect of acidosis from lack of oxygen causes interruption at several critical steps. First, glucose cannot gain access to the cellular interior. Next, phosphofructokinase, the enzyme which catalyzes the conversion of fructose, 6, monophosphate, to fructose 1,6, diphosphate is rendered inactive.
To bypass these metabolic bottlenecks, fructose 1,6 diphosphate can be injected in amounts exceeding substantially that which could be available in the natural state. This sugar, if metabolized to lactic acid, will produce 4 molecules of ATP without the requirement of oxygen. Besides, the fructose molecule unlike glucose does not require energy to cross the cell membrane and is not dependent upon the action of insulin and it enters above the PFK enzyme level which has been damaged during the ischemic process. Therefore, the addition of fructose 1,6 diphosphate appears to be a significant step in by-passing this and obtaining temporary energy to sustain ischemic tissues, perhaps even the whole organism over a limited time while it is being infused. It may produce from 20 to 80 percent of the energy required for a given tissue. In a number of experiments which have been carried out by Dr. Angel Markov, in the Department of Medicine at the University of Mississippi, it has been shown repeatedly that the material is useful when given in hemorrhagic shock, experimental myocardial infarction, and other conditions where tissue is without oxygen. In Dr. James W. Jones' experiments, it has appeared that this material is very useful in metabolism of the heart. The research gives an evidence that the material could be useful in treating patients with the following disorders:
1. Hemorrhagic shock; PA0 2. Cardiogenic shock; PA0 3. Cyanide poisoning and any poisoning of oxidative metabolism; PA0 4. Myocardial preservation; PA0 5. Therapeutic agent after myocardial infarction; PA0 6. During respiratory failure with low blood oxygen levels; PA0 7. During operative procedures as a protective agent against unforeseen catastrophic hypotension or hypoxia; PA0 8. As a preservative agent for transplantation organs such as kidney, liver, heart, etc.; PA0 9. As an agent to enhance drugs it would be given as chemotherapeutic agents to destroy a tumor cell; PA0 10. Sickle-Cell anemia; PA0 11. Reversal barbituate overdose; PA0 12. Blood preservation; PA0 13. As an agent for treating disorders in white blood cells' phagocytosis; and PA0 14. Endotoxin shock.
In the experiments regarding an irreversible hemorrhagic shock, results were obtained in the dog model using Wigger's modified technique. We compared a group of animals receiving IV administration of fructose-1,6-diphosphate (FDP) to a group receiving equimolar glucose.
The metabolic changes resulting from generalized tissue hypoxia revealed alteration in the acid base balance and increased lactic acid concentration in blood reflecting the relatively anaerobic character of the metabolism. The rate of anaerobic glycolysis for different tissue appears to be a direct function of the severity of hypotension and the severe hypotension could accelerate glycolysis in the heart and other organs by 100% when compared to control. Although there is an initial increase of anaerobic glycolysis after a certain period, its rate begins to decline due to progressively increasing acidosis secondary to increased lactemia.
The phosphorylation of fructose-6-phosphate is an important control point in the Embden-Meyerhof pathway. Phosphofructokinase (PFK) is a multivalent enzyme which catalyzes this rate limiting reaction. PFK is stimulated by ADP, AMP and FDP and inhibited by ATP, citrate and acidosis. It is already known that the ischemic inhibition of glycolysis is due to inactivation of PFK by progressively increasing intracellular acidosis. As in shock severe metabolic acidosis takes place, it is reasonable to assume that PFK is inactivated. This is substantiated by the observation that in hemorrhagic shock plasma lactate initially increases very rapidly and then lactate production appears to decline. In our experiments the plasma lactate measured at 2 hrs 45 min after the onset of hypotension in the controls was found to be 76.+-.14 mg %, while in dogs treated with FDP that concentration was in the order of 124.+-.16 mg %.
Our experiments also gave evidence that serious cardiac failure (secondary to impaired energy production) occurs approximately at the time when hemorrhagic shock becomes irreversible and it is probably responsible for the fatal outcome of the condition. It is realized that the pathogenesis of hemorrhagic shock is a complex phenomenon in which every structural unit in the entire organism is affected (to different degrees). Yet, the circulatory weak spot contributing to the irreversibility of the hemorrhagic syndrome appears to be the heart itself. Forty years ago Wiggers realized that in the course of hemorrhagic shock, a deficit in coronary blood flow that arises could be implicated as a major cause for the irreversibility of the condition.
In control animals during the late course of the hemorrhagic shock changes were observed in metabolism in the endocardium taking place to be similar to those observed in acute myocardial ischemia. These are manifested by ST segment elevation which occurred in all non-treated animals and depletion of ATP and CP in the endocardium to the same degree as found in acute myocardial infarction.
To allow for a broader and more complete interpretation of the results, a small paragraph will be devoted to the ability of FDP to cross the cellular membranes. Although the actual mechanism of trans-membranous passage of FDP is not known, there is direct and indirect evidence which substantiate that it crosses through cellular membranes. These data are consistent with the modern concept of the membrane which is capable of transferring high energy substrate through a series of coupled reactions. From experimental data, indirect evidence substantiates the assumption that FDP crosses the cellular membrane in different tissues in animals and man. In isolated organs, for example, when FDP is added perfusate, rabbit ileum increases the force of contraction, while glucose-6-phosphate, fructose-6-phosphate or fructose and inorganic phosphate fail to produce the same effect. This effect of FDP on rabbit ileum can be correlated with the increased availability of ATP and the regulatory effect of FDP on PFK, on pyruvate kinase and in inhibition of 6-phosphogluconate dehydrogenase. As all of these enzymes are in the cytoplasm, FDP must cross the cellular membrane in order for such an effect to be observed. Incubation of erythrocytes with 5% FDP causes a large increase in their ATP and 2-3 DPG content. Equimolar glucose, glucose-6-phosphate, fructose-6-phosphate, fructose and inorganic phosphate failed to increase the ATP content in the erythrocytes incubated under the same conditions. Intracardiac administration of FDP in rat causes five times higher concentration of FDP in the liver than in plasma 10 min after administration. Intravenous administration in dogs of 5 g of FDP over 10 min causes the plasms lactic acid (measured at one hour) to increase 21/2 times (from 6.13.+-.1.7 to 16.45.+-.2.16 mg %). In other experiments we have found that IV administration of FDP causes an increase of ATP, lactate, and FDP in all tissues that we have studied, as well as increases in the trioses in the Embden-Meyerhof pathway.
We attempted to remove the inhibition of PFK, hence glycolysis, in the shock model described by intravenous administration of FDP, thus increasing energy production, preventing cardiac damage and improving survival. Fructose diphosphate in the described shock model reduced mortality to zero, prevented electrocardiographic ischemic changes and increased significantly the ATP and CP in the endocardium. These changes were asspociated with accelerated glycolysis seen by increased plasma lactate and myocardial tissue lactate.
From an energetic point of view the advantage of using FDP as the initial substrate is that the net yield of the anaerobic metabolism of one mole of glucose is 2 ATP, while if one mole of FDP is metabolized in the same conditions it will produce 4 ATP because there is no phosphorylation of glucose and fructose-6-phosphate, reactions which require utilization of ATP. On the other hand, while doubling the quantity of ATP produced, lactate production remains the same as though one mole of glucose had been metabolized. It is obvious that in order to obtain results in the direction of making usable energy from FDP, it is necessary to administer a large quantity. (The LD.sub.50 of FDP when administered 500 mg/min in dogs is approximately 5.8-6 g/kg.)
Another important metabolic effect of FDP is that it causes a substantial increase of ATP and 2-3 DPG in the erythrocytes. This increase of 2-3 DPG is of importance for the oxygen exchange between hemoglobin and tissue. It may also contribute in part to energy production derived from the oxidative metabolism by increasing the oxygen availability to tissues in such a low flow state.
The significance of the present invention is not only limited to the successful treatment of experimental irreversible hemorrhagic shock by increasing metabolic activity of glycolysis, but as well it proposes a unifying concept for understanding the pathophysiological mechanism of shock. There is no doubt that the nature of the initial insult is irrelevant to the genesis of shock, but what is important is the fact that the organism as a whole integral unit shares with its constituents (organs and cells) a common energy deficit. In the early stages of any etiologic type of shock there is inadequate oxygen transport and supply to tissue. Hence, there is an energy deficit. Every system, organ and individual cell responds to this decrease in energy supply according to their entropic state. As the biological system is an open system, any deficit in free energy would be manifested by increasing the disorder of the system and as in shock the vicious cycle is initiated which leads the system to higher and higher degrees of disorder. The final stage of this intracellular disorder is the achievement of the same entropic state between "milieu exterieur" and "milieu interieur"--defined in biological terms as death.
It is thus an object of the present invention to provide a method of treating patients during a hemorrhagic shock by fructose-1,6-diphosphate (FDP).
It is another object of the present invention to provide a method of treating patients in cardiogenic shock with FDP.
It is a further object of the present invention to provide a method of treating patients in case of cyanide poisoning by FDP.
It is another object of the present invention to provide a method of myocardial preservation by treating a patient with FDP.
It is a further object of the present invention to provide a method of treating patients after myocardial infarction by using FDP as a therapeutic agent.
It is still another object of the present invention to provide a method of treating patients with FDP during respiratory failure with low blood oxygen levels.
It is a further object of the present invention to provide a method of introducing FDP during operative procedures so that it might be a protective agent against unforeseen catastrophic hypotension or hypoxia.
It is still another object of the present invention to provide a method of preservation of transplanted organs using FDP as a preservative agent.
Still another object of the present invention is to provide a method of treating cancer patients by using FDP as a possible means of causing damage to the tumor cells and using it as a chemotherapeutic agent to enhance drugs in destroying tumor cells.
It is further an object of the present invention to provide a method of treating patients with FDP during a sickel cell crisis.
Still another object of the present invention is to provide a method of treating patients having a reversal barbituate overdose.
Another object of the present invention is to provide a method of using FDP for blood preservation.
It is another object of the present invention to provide a method of treating patients with disorders in white blood cells' phagocytosis using FDP.
Finally, another object of the present invention is to provide a method of treating patients with FDP during an endotoxin shock.